DNA mismatch repair (MMR) is essential for maintaining the integrity of mammalian genomes by removing misincorporated nucleotides that result from erroneous replication and by mediating a DNA damage response (DDR) after exposure to genotoxic agents. Mutations in mammalian MMR genes are causative in hereditary non-polyposis colorectal cancer /Lynch syndrome (HNPCC/LS) and many sporadic cancers. Importantly, loss of MMR not only increases genomic mutation rates but also the resistance of tumors to conventional chemotherapeutic agents. Our research program focuses on elucidating the different biological functions of the individual MMR genes involved in mismatch recognition and excision and assessing their importance for tumor suppression and the response to chemotherapeutic agents. In the past funding period, we determined that MMR-dependent DDR is essential for tumor suppression and the sensitivity of tumors to chemotherapeutic treatment. In new preliminary studies, we have modeled the pathogenic S144I HNPCC/LS mutation located in the MSH6-PWWP protein interaction domain, in knock-in mice. PWWP domains have been shown to directly interact with methylated lysines in histones suggesting a novel interaction of MMR proteins with chromatin. The MSH6S144I mutation is unique, because in contrast to all other known MMR mutations, it only impairs the DDR function but does not affect the repair of mismatched bases. This mutation will allow us for the first time to unequivocally test the hypothesis that MMR-dependent DDR function is essential for suppression of diet and carcinogen-induced tumorigenesis and also identify novel MSH6-chromatin interactions. We have also found in VCMsh2loxP knockout mice that were developed by us and serve as excellent preclinical models of HNPCC/LS, that treatment of intestinal tumors with rapamycin results in dramatic tumor regression and increased life span, suggesting that rapamycin treatment may have clinical impact in being specifically effective in the treatment of MMR-deficient tumors. To determine the molecular mechanisms underlying the regression of MMR-deficient intestinal tumors in response to rapamycin treatment we will test the idea that the accumulation of rapamycin- induced oxidative DNA damage and autophagy cooperate to cause selective sensitivity in MMR-deficient intestinal tumors. Finally, we propose to determine the importance of MMR in intestinal tumor stem cells for the sensitivity or resistance of intestinal tumors to chemotherapeutic treatment.